The human dental plaque biofilm is a physically and chemically complex environment that is inhabited by a many bacterial species, including Streptococcus mutans, which is regarded as the primary etiological cause of human dental caries. S. mutans has a number of behaviors that give rise to its virulence, and it regulates and activates these behaviors through the use of chemical cues received from its environment. However, the local environment of S. mutans at different locations within a biofilm may be chemically and physically different, subject to substantial variation in pH, oxygen concentration and nutrient availability, as well as different balances of colonizing species and different concentrations of the small molecules that allow the bacteria to communicate and regulate virulence behaviors. This project seeks to determine how this diversity of microenvironments affects the centrally important S. mutans virulence behavior known as genetic competence. Competence is the ability to take up extracellular DNA, and the genetic network that regulates competence in S. mutans is closely intertwined with the mechanisms that regulate virtually every other cariogenic trait of the organism. The competence genes are extremely sensitive to environmental conditions and to the nature of the chemical signals received. Expression of competence genes across a population of cells can be uniform or can involve activation of only a subset of the bacteria. Therefore a broad scientific goal is to understand how S. mutans processes or interprets environmental signals to regulate competence and other virulence behaviors, how microenvironments in the biofilm affect this processing, and how competence genes are activated at the cell-to-cell level throughout an oral biofilm. This project will focus o identifying and understanding the genetic switches that control S. mutans competence, exploring how pH, oxygen concentration, and carbohydrate availability affect the regulation of competence by peptide signals, and studying and modeling the ways that competence is regulated by peptide signals inside an S. mutans biofilm. The project will accomplish these goals through a novel microfluidic, single-cell approach. This method allows multiple, well-defined environmental inputs to be supplied to subpopulations of S. mutans cells while the profile of competence gene activation across those subpopulations is measured and modeled quantitatively. The project will yield detailed information about how S. mutans competence is spatially distributed in an oral biofilm and what kinds of chemical conditions trigger this and related virulence behaviors. Success in achieving these objectives will advance research in human oral health by improving the understanding of how Streptococcus mutans causes dental disease.